Myosin from macrophages is thought to play an essential role in cell motility and phagocytosis, though its specific interaction with actin filaments (actin-activation of the MgATPase activity of myosin). In vitro, macrophage myosin is activated by actin in proportion to the extent of phosphorylation of a single site on ts 20 KD light chain. In living macrophages both the 20 KD light chain and the heavy chain of myosin are phosphorylated. Heavy chain phosphorylation is mediated by a kinase that is distinct from the kinase which phosphorylates the light chain. These observations strongly suggest that the activity of macrophage myosin is regulated by two independent, reversible phosphorylations: one related to the 20 KD light chain and one related to the heavy chain. The objective of this proposal are to elucidate the mechanism and significance of heavy chain phosphorylation by: a) characterization of the phosphorylated sites; b) characterization of the kinase and phosphatase that act upon the heavy chain; and c) determination of the effects of heavy chain phosphorylation on myosin assembly, actin binding, actin-activation, ATPase activity, and myosin-myosin cooperativity. These experiments should advance our knowledge of the regulation of myosin, and hence of cell motility, in macrophages and potentially in other cell types as well.